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Cancer Therapy: Preclinical

Lenalidomide Enhances Immune CheckpointBlockade-Induced Immune Response in MultipleMyelomaG€ull€u G€org€un1, Mehmet K. Samur1,2, Kristen B. Cowens1, Steven Paula1, Giada Bianchi1,Julie E. Anderson1, Randie E.White1, Ahaana Singh1, Hiroto Ohguchi1, Rikio Suzuki1,Shohei Kikuchi1, Takeshi Harada1, Teru Hideshima1, Yu-Tzu Tai1, Jacob P. Laubach1,Noopur Raje3, Florence Magrangeas4,5, Stephane Minvielle4,5, Herve Avet-Loiseau6,Nikhil C. Munshi1,7, David M. Dorfman8, Paul G. Richardson1, and Kenneth C. Anderson1

Abstract

Purpose: PD-1/PD-L1 signaling promotes tumor growth whileinhibiting effector cell–mediated antitumor immune responses.Here, we assessed the impact of single and dual blockade of PD-1/PD-L1, alone or in combination with lenalidomide, on accessoryand immune cell function as well as multiple myeloma cellgrowth in the bone marrow (BM) milieu.

Experimental Design: Surface expression of PD-1 on immuneeffector cells, and PD-L1 expression on CD138þ multiple mye-loma cells and myeloid-derived suppressor cells (MDSC) weredetermined in BM from newly diagnosed (ND) multiple myelo-ma and relapsed/refractory (RR) multiple myeloma versushealthy donor (HD). We defined the impact of single and dualblockade of PD-1/PD-L1, alone and with lenalidomide, on auto-logous anti–multiple myeloma immune response and tumor cellgrowth.

Results: Both ND and RR patient multiple myeloma cells haveincreased PD-L1 mRNA and surface expression compared withHD. There is also a significant increase in PD-1 expression on

effector cells in multiple myeloma. Importantly, PD-1/PD-L1blockade abrogates BM stromal cell (BMSC)-induced multiplemyeloma growth, and combined blockade of PD-1/PD-L1 withlenalidomide further inhibits BMSC-induced tumor growth.These effects are associated with induction of intracellular expres-sion of IFNg and granzyme B in effector cells. Importantly, PD-L1expression in multiple myeloma is higher on MDSC than onantigen-presenting cells, and PD-1/PD-L1 blockade inhibitsMDSC-mediated multiple myeloma growth. Finally, lenalido-mide with PD-1/PD-L1 blockade inhibits MDSC-mediatedimmune suppression.

Conclusions: Our data therefore demonstrate that checkpointsignaling plays an important role in providing the tumor-pro-moting, immune-suppressive microenvironment in multiplemyeloma, and that PD-1/PD-L1 blockade induces anti–multiplemyeloma immune response that can be enhanced by lenalido-mide, providing the framework for clinical evaluation of combi-nation therapy. Clin Cancer Res; 21(20); 4607–18. �2015 AACR.

IntroductionMultiple myeloma is a clonal B-cell malignancy associated

with a monoclonal (M) protein in blood and/or urine, bonelesions, and immunodeficiency. It usually evolves from mono-

clonal gammopathy of undetermined significance (MGUS),with low levels of plasmacytosis and M protein without osteo-lytic lesions, anemia, hypercalcemia, and renal failure (1).Multiple myeloma is characterized by genetic signatures,including frequent translocations into the immunoglobulinheavy chain switch region (IgH), oncogenes, and abnormalchromosome number (2, 3). Most patients with translocationshave non-hyperdiploid chromosome number (NHMM), whilethose patients lacking IgH translocations have hyperdiploidchromosome number (HMM) with trisomies of chromosomes3,5,7,9,11,15,19, and 21. Importantly, patients with hyperdi-ploid multiple myeloma have a better outcome with prolongedsurvival (4, 5).

Advances in multiple myeloma biology have established thatthe bidirectional interaction between multiple myeloma cells,bone marrow stroma cells (BMSC), extracellular matrix, andaccessory cells can induce autocrine and paracrine signaling thatregulates tumor development and growth on the one hand, whiletransforming the BM microenvironment into an immune-sup-pressivemilieu on the other (6, 7).We andothers have extensivelystudied the impact of the interaction between BMSC andmultiplemyeloma cells on pathogenesis and cell adhesion mediated-drugresistance (CAM-DR) in order to identify and validate new

1Department of Medical Oncology, Dana-Farber Cancer Institute, Har-vard Medical School, Boston, Massachusetts. 2Department of Biosta-tistics and Computational Biology, Harvard School of Public Health,Boston, Massachusetts. 3Massachusetts General Hospital, Boston,Massachusetts. 4Inserm UMR892, CNRS 6299, Universit�e de Nantes,Nantes, France. 5Centre Hospitalier Universitaire de Nantes, Unit�eMixte deGenomique duCancer, Nantes, France. 6Unite deGenomiqueduMyelome,CHURangueil,Toulouse, France. 7BostonVAHealth CareSystem, Boston, Massachusetts. 8Department of Pathology, Brighamand Women's Hospital, Boston, Massachusetts.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Authors: G€ull€u G€org€un, Dana-Farber Cancer Institute, Depart-ment of Medical Oncology, 450 Brookline Ave, Room M557, Boston, MA, 02215.Phone: 617-632-6553; Fax: 617-632-2140; E-mail: [email protected];and Kenneth C. Anderson, E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-15-0200

�2015 American Association for Cancer Research.

ClinicalCancerResearch

www.aacrjournals.org 4607

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targeted therapeutics (1). Immunomodulatory drugs thalido-mide and lenalidomide, and proteasome inhibitor bortezomibare novel agents which target the tumor cell in its microenviron-ment and can overcome CAM-DR; they have been rapidly inte-grated into multiple myeloma treatment, resulting in at least a 2-to 3-fold prolongation of median survival (8–10). Even thoughthese novel drugs have transformed the treatment paradigm andpatient outcome, most multiple myeloma relapses due to min-imal residual disease and drug resistance (11). Generation ofmore effective therapeutic strategies may therefore not onlyrequire targeting the tumor and stroma, but also overcomingblockade of antitumor immune response. Tumor-associatedimmune suppressor cells such as regulatory T cells (Treg) andmyeloid-derived suppressor cells (MDSC) can effectively blockantitumor immune responses, representing an important obstaclefor immunotherapy. We have recently assessed the presence,frequency, and functional characteristics of MDSC in patientswith newly diagnosed (ND-MM), responsive multiple myeloma,and relapsed, refractory multiple myeloma (RR-MM) comparedwith healthy donor (HD), and identified an increased MDSCpopulation (CD11bþCD14�HLA-DR�/lowCD33þCD15þ) withtumor-promoting and immune-suppressive activity in both theperipheral blood (PB) and BM of multiple myeloma patients.Moreover, we have shown that lenalidomide does not targetMDSC in the BM milieu (12).

Programmed cell death-1 (PD-1, CD279), a member of theCD28 receptor family, and its ligands either PD-L1 (B7-H1,CD274) or PD-L2 (B7-DC, CD273), play a fundamental role intumor immune escape by inhibiting immune effector functions.PD-1 gene is encoded on chromosome 2, and PD-L1 gene is onchromosome 9. PD-1 expression is induced on antigen activated Tcells and exhausted T cells and B cells; PD-L1 is mainly expressedby antigen-presenting cells (APC) and various nonhematopoieticcells; and PD-L2 is found on hematopoietic cells, includingdendritic cells and macrophages (13). Recent studies in solidtumors have demonstrated that expression of PD-L1 is signifi-

cantly increased and associated with progressive disease in lungcancer, breast cancer, renal cell cancer, colorectal cancer, gastriccancer, esophageal cancer, and pancreatic cancer (7, 8, 14–21).Most importantly, remarkable responses have been observed toPD-1 blockade in malignant melanoma, leading to recent FDAapproval of PD-1 monoclonal antibody therapies. To date,increased PD-L1 expression has been shown inmultiplemyelomacells compared with HD plasma cells (13, 22–26), and increasedPD-1 expression has been demonstrated on CD4T cells in mul-tiple myeloma (11, 13, 22, 24, 25, 27). Because PD-1/PD-L1signaling promotes tumor growth while inhibiting effector cell–mediated antitumor immune response, we here assessed theimpact of single and dual blockade of PD-1/PD-L1 signaling,alone or in combinationwith lenalidomide, on accessory (MDSC,BMSC) and immune cell (CD4T cells, CD8T cells, NK cells, NKTcells, and monocytes/macrophages) function, as well as multiplemyeloma cell growth, in the BMmilieu. Our studies provided theframework for targeting PD-1 and PD-L1 in combination withlenalidomide to inhibit tumor cell growth and restore immunefunction in multiple myeloma.

Materials and MethodsCell isolation

Heparinized venous blood samples and/or aspirates of BMfrom patients with ND-MM (n ¼ 6) or RR-MM (n ¼ 10) andhealthy donors (HD, n ¼ 10) were obtained after writteninformed consent per the Declaration of Helsinki and approvalby the Institutional Review Board of the Dana-Farber CancerInstitute (Boston, MA).

Cell linesMM1.S, U266, and H929 multiple myeloma cells were pur-

chased from ATCC; plasma cell leukemia (PCL) cells OPM1 andOPM2 were provided by Dr. Edward Thompson (University ofTexas Medical Branch, Galveston, TX). Cell lines have been testedand authenticated by STRDNA fingerprinting analysis (MolecularDiagnostic Laboratory, DFCI), and used within 3 months afterthawing. All cell lines were maintained in RPMI-1640 (Bio Whit-taker) containing 10% FBS, 100 U/mL penicillin, and 100 mg/mLstreptomycin (Life Technologies).

Reagents and compoundsFunctional grade PD-1 and PD-L1 blocking antibodies, anti-

human PD-1 (clone J116) and anti-human PD-L1 (clone MIH1),were obtained from eBiosciences. Immunomodulatory drug lena-lidomide (10 mmol/L) was dissolved in DMSO and stored at�20�C. Anti-CD3 and anti-CD28 MAbs (10 mg/mL; Becton Dick-inson Biosciences) were used to stimulate cells.

Cell phenotypingCell surface expression of PD-1 (CD279) on CD4þT cells,

CD8þT cells, CD56þNK cells and CD3þCD8þCD56þNKT cells,and PD-L1 (CD274) on CD138þ multiple myeloma cells,CD14þ monocytes/macrophages, CD11bþCD14þHLA-DRþ

APCs, CD11bþCD14�HLA-DR�/lowCD33þCD15þ nMDSC,and CD11bþCD14þHLA-DR�/low mMDSC was determined onPBMCs or BMMCs from multiple myeloma patients or healthydonors by multiparameter flow-cytometric analysis. Cells werestained with CD11b APCCy7, CD14 Pacific blue, HLA-DRPECy7, CD33 PECy5, and CD15 FITC conjugated MAbs (BD

Translational Relevance

The interaction of tumor cells with their surrounding acces-sory cells and extracellularmatrix provides a tumor-promotingenvironment while suppressing immune response. Recentstudies in solid tumors have demonstrated that programmeddeath-1 (PD-1) signaling plays an important role in tumor-induced immune suppression and conversely, that blockadeof PD-1/PD-L1 by therapeutic antibodies restores antitumorimmune response. Remarkable responses have been observedto PD-1 blockade in malignant melanoma, leading to recentFDA approval of anti–PD-1 antibody therapies. Here, weassessed the impact of single and dual blockade of PD-1/PD-L1, alone or in combination with lenalidomide, on acces-sory and immune cell function, as well as on multiple mye-loma cell growth in the BM milieu. Our study demonstratesthat PD-1/PD-L1blockade can induce anti–multiplemyelomaimmune responses, which are enhanced by lenalidomide. Ourstudies provide the preclinical rationale for evaluation ofcombined PD-1/PD-L1 blockade with lenalidomide to inhibittumor cell growth, restore host immune function, andimprove patient outcome in multiple myeloma.

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Clin Cancer Res; 21(20) October 15, 2015 Clinical Cancer Research4608




Figure 1.Increased frequency of PD-1 and PD-L1 expression in multiple myeloma BM microenvironment. Cell surface expression of PD-1 (CD279) and PD-L1 (CD274) isshown on CD138þ multiple myeloma cells, immune effector cells, and immune-suppressive MDSC in ND-MM and RR-MM. A, cell surface expression of PD-L1is quantitated onpatientmultiplemyelomacells obtained frompatientswithND-MM (n¼6) andRR-MM (n¼ 10), and comparedwith healthydonor plasma cells (HD,n ¼ 3). Data represent percentage of PD-L1–expressing CD138þ multiple myeloma cells. Representative histogram plots of PD-L1 expression (red) relative tocontrol (gray) is shownon agated population of CD138þplasma cells. Top panel demonstrates PD-L1 expression onmultiplemyeloma cell lines (MM1.S, OPM2, H929),and bottom panel represents PD-L1 expression on BM CD138þ plasma cells from patients with ND-MM and RR-MM, as well as HD-BM. B, cell surface expressionof PD-1 is quantitated on immune effector cells (CD4T cells, CD8T cells, NK cells, and NKT cells) from patients with ND-MM (n ¼ 6) and RR-MM (n ¼ 10)compared with healthy donors (HD, n¼ 10). Data represent percentage of PD-1–coexpressing CD4T cells (top left), CD8T cells (top right), NK cells (bottom left), andNKT cells (bottom right) in BM of patients with ND-MM and RR-MM compared with HD-PBMC. Representative histogram plots of PD-1 expression (blue)versus control (gray) on BM immune effector cells: CD4T cells, CD8T cells, NK cells, and NKT cellswith gating strategy are shown bymultiparameter dot plots (right).C, the frequency of PD-L1 cell surface expression is shown in monocytic MDSC (mMDSC) and neutrophilic MDSC (nMDSC) compared with APCs from BM of patientswith ND-MM (left) and RR-MM (right). Representative flow-cytometric histogram of PD-L1 expression (red) versus control (gray) on MDSC in healthy donorand RR-MM BM (right) with gating strategy for mMDSC and nMDSc is shown by multiparameter dot plots (right). � , P < 0.05.

Lenalidomide in Combination with Checkpoint Blockade in Multiple Myeloma

www.aacrjournals.org Clin Cancer Res; 21(20) October 15, 2015 4609




Biosciences) for MDSC; as well as CD138 APC for multiplemyeloma cells, and CD4 PE-Cy5, CD8 PE-Cy7, CD56 FITC foreffector cells (BD Biosciences).

Intracellular cytokine analysisAutologous effector cells and CD138þ multiple myeloma cells

or MDSCs were cocultured in the absence or presence of anti-human PD-1 (10 mg/mL) and anti-human PD-L1 blocking Ab (10mg/mL) alone or in combination with or without addition oflenalidomide (1 mmol/L) for 16 hours to 4 days. Of note, 5 mg/mLof brefeldin-A solution (eBiosciences) was added during the last 2hours of incubation. Cells were then fixed in 4% paraformalde-hyde-PBS and stained with PE-conjugated IFNg and FITC-conju-gated granzyme B (Gzm-B)MAbs (BectonDickinson Biosciences)in permeabilization buffer (0.5% saponin-PBS). Intracytoplasmiccytokines in T cells, NK cells, NKT cells, and monocytes/macro-phages was detected by flow cytometry using BD-LSR Fortessa(Becton Dickinson Biosciences) and analyzed using FlowJo soft-ware (TreeStar).

Cell proliferation assayCD138þmultiple myeloma cells were isolated by FACSAria IIu

sorter, labeled with CFSE, and cultured either alone or with BMSC

generated from BM aspirates of multiple myeloma patients, orautologous MDSC, with or without anti-human PD-1 and anti-human PD-L1 Ab alone or in combination for 24 hours to 4 days(2:1 ratio). Multiple myeloma cell growth was measured follow-ing propidium iodide addition (PI, 1 mg/mL) by CFSE/PI flow-cytometric analysis using BD-LSR Fortessa (Becton DickinsonBiosciences), and data were analyzed using FlowJo software(TreeStar).

MDSC and CD3þT cells were isolated from PB or BMaspirates of multiple myeloma patients by FACS-sorting,and MDSC were cocultured for 4 days with CFSE-labeledautologous T cells (MDSC:T cell ratio 1:4) in the absence orpresence of checkpoint blockade antibodies with lenalidomide(1 mmol/L).

Cytotoxicity assayCD138þ multiple myeloma cells and autologous effector

cells (CD3T cells and NK cells) were isolated by FACS sortingfrom multiple myeloma BM. CD138þ multiple myeloma cellswere labeled with CFSE and cultured with each effector cellpopulation in the absence or presence of anti–PD-1 or anti–PD-L1, alone or in combination, for 4 hours. PI (1 mg/mL) wasadded before analysis. Apoptotic/dead multiple myeloma cells

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Figure 2.Checkpoint blockade overcomes BM stroma-mediated multiple myeloma growth. PD-1/PD-L1 signaling role in the bidirectional interaction between multiplemyeloma cells and BM stromal cells (BMSC) is shown in coculture of BMSC andmultiple myeloma cells. A, BMSC effect on PD-L1 expression inmultiple myeloma cellsis demonstrated by multiparameter flow-cytometric analysis of multiple myeloma cell–BMSC cocultures. Representative histogram plot for PD-L1 expression inCD138þ multiple myeloma cell population (red) versus control (gray) is demonstrated in multiple myeloma cell line (H929) alone and cultured with BMSC. B,checkpoint blockade effect on BMSC-mediated multiple myeloma growth is demonstrated by CFSE-flow analysis in cocultures of CD138þ multiple myeloma cellsfrom patient with RR-MMwith BMSC. Shown are multiparameter dot plots for multiple myeloma cells alone and cultured with BMSCwith or without single and dualblockade of PD-1 and PD-L1. CD138þCFSElow cell population represents live/growth multiple myeloma cells (large gated box), and CD138þCFSEhigh cell populationrepresents non-dividing/dead multiple myeloma cells (small gated box).

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were identified as CD138þCFSEþPIþ by multiparameter flow-cytometric analysis using BD-LSR Fortessa (Becton DickinsonBiosciences), and data were analyzed using FlowJo software(TreeStar).

To determine the effect of checkpoint blockade with lenalido-mide on anti–multiple myeloma cytotoxicity in the BM, BMmononuclear cells (BMMC) from patients with RR-MM werelabeled with CFSE and cultured in the absence or presence ofanti–PD-1 (10 mg/mL) and anti–PD-L1 (10 mg/mL), alone or in

combination, orwith the addition of lenalidomide (1mmol/L). PI(1 mg/mL) was added before analysis. Immune complex withApoptotic/dead multiple myeloma cells was identified asCD138þCFSEþPIþ by multiparameter flow-cytometric analysisusing BD-LSR Fortessa (Becton Dickinson Biosciences), and datawere analyzed using FlowJo software (TreeStar). Minimum of10,000 live events per sample were acquired using multiparam-eter flow cytometry.

Statistical analysisAll in vitro experiments were performed in triplicate and repeat-

ed at least three times. Statistical significance was determined bynonparametric t test, two-tailed distribution, with minimal sig-nificance level �, P < 0.05. Statistical analysis was performed usingGraphPad Prism (v6) software.

ResultsIncreased PD-L1 gene expression in multiple myeloma

We first assessed expression and frequency of PD-L1 gene inthe BM CD138þ multiple myeloma cells from patients withmultiple myeloma at diagnosis [ND-MM; Intergroupe Franco-phone du Myelome (IFM), n ¼ 170] versus normal BM plasmacells (HD, n ¼ 6; Supplementary Fig. S1A). Exon array profilinganalysis of CD138þ plasma cells demonstrated that there was asignificant increase in PD-L1 mRNA expression in multiplemyeloma cells from patients with ND-MM compared with HD(P ¼ 0.0064). Furthermore, 45% patients had increased copynumber of PD-L1 gene in their tumor clone (SupplementaryFig. S1B). Expression and copy number of PD-L1 gene weresignificantly correlated in clonal multiple myeloma cells (Pear-son correlation, R2 ¼ 0.37 and P ¼ 5.5e-07; Supplementary Fig.S1C). Because PD-L1 gene is encoded on chromosome 9, wenext compared PD-L1 gene expression among normal, hyper-diploid multiple myeloma (HMM) and non-hyperdiploidmultiple myeloma (NHMM) groups. PD-L1 gene expressionwas significantly upregulated in NHMM (P ¼ 0.03), and evenmore highly expressed in HMM (P ¼ 0.0007), comparedwith normal plasma cells. In addition, there was a significantdifference between HMM and NHMM subgroups (P ¼ 1.75e-09; Supplementary Fig. S1D). In contrast, there was no signif-icant association between PD-L1 expression and chromosomalabnormalities in multiple myeloma, including t(4;14), del(17p), del (1q), t(11;14), del(13), and t(14;16; data notshown).

Increased frequency of PD-1 and PD-L1 surface expression inmultiple myeloma BM microenvironment

We next assessed surface expression of PD-L1 by multipa-rameter flow cytometry in BM CD138þ multiple myeloma cellsfrom patients with ND-MM (n ¼ 6), RR-MM (n ¼ 10), andnormal plasma cells (n ¼ 3); as well as in a panel of multiplemyeloma and PCL cell lines (MM1.S, OPM1, OPM2, U266, andH929; Fig. 1). PD-L1 surface expression was significantlyincreased in multiple myeloma cells from patients with ND-MM (mean ¼ 16.5 � 6.5) and RR-MM (mean ¼ 26.6 � 8.2)compared with normal plasma cells (mean ¼ 4.7 � 1.8; P <0.05; Fig. 1A, left and bottom right), and was detectable only inMM1.S, OPM2, and H929 multiple myeloma cell lines (Fig. 1A,top right). There was no significant expression of PD-L2 onmultiple myeloma cells (data not shown).

Figure 3.Checkpoint blockade enhances immune effector cell–mediated anti–multiple myeloma responses in multiple myeloma BM. A, impact ofcheckpoint blockade on immune effector cell–mediated anti–multiplemyeloma response is demonstrated by a multiparameter CFSE/PI flow-cytometric analysis. CFSE-labeled target CD138þ multiple myeloma cellsand autologous immune effector cells (CD3T cells and NK cells) werecultured with or without anti–PD-1 and anti–PD-L1, alone or incombination. Effector cell–mediated cytotoxicity was determined byCD138þCFSEþPIþ apoptotic/dead multiple myeloma cells. Fold changeis relative to control (effector cell–mediated multiple myelomacytotoxicity without checkpoint blockade). Impact of checkpoint blockadeon effector cytokines IFNg (B) and Gzm-B (C) mediating cytotoxicityagainst multiple myeloma is shown in effector cells in cocultures ofCD138þ multiple myeloma cells and autologous effector cells. � , P < 0.05.






Figure 4.Checkpoint blockade partially reverses MDSC-mediated multiple myeloma growth and immune suppression in multiple myeloma BM. A, impact of checkpointblockade on MDSC-mediated tumor growth is demonstrated in the BM of patients with RR-MM by CFSE-flow cytometric analysis. CFSE-labeled CD138þ

multiple myeloma cells and autologous MDSC were cultured in the absence or presence of anti–PD-1 and anti–PD-L1, alone or in combination. Viability/growthof CD138þ multiple myeloma cells (CD138þCFSElow) is shown by representative histogram plots. B, effect of checkpoint blockade on MDSC-mediated immunesuppression is shown by intracellular effector cytokine analysis in RR-MM BM. Autologous T cells cultured either alone or with mMDSC and nMDSC in theabsence or presence of anti–PD-1 and anti–PD-L1, alone or in combination. (Continued on the following page.)

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We next evaluated surface expression of PD-1 in immuneeffector cells in both BM and peripheral blood of patients withactive multiple myeloma using multiparameter flow cytometry(Fig. 1B). Even though CD4T cells from several patients with ND-MM and RR-MM expressed high levels of PD-1, there was nosignificant difference in PD-1 expression on CD4T cells in mul-tiple myeloma BM, either ND-MM (mean ¼ 10.8 � 3.4) or RR-MM(mean¼12�2.8), comparedwithnormalCD4T cells (mean¼ 10.2� 1.3; Fig. 1B, top left). In contrast, there was a significantincrease in expression of PD-1 onCD8T cells in ND-MM (mean¼10.4 � 1.7) and RR-MM (mean ¼ 9.4 � 2.3) compared withnormal CD8T cells (mean ¼ 6.1 � 0.6; Fig. 1B, top right).Likewise, PD-1 expression was higher in BM NK cells in patientswith ND-MM (mean ¼ 7.9 � 1.4) and RR-MM (mean ¼ 14.2 �3.2) compared with normal NK cells (mean ¼ 1.6 � 0.2; Fig. 1B,bottom left). In contrast, PD-1 expression was significantly lowerin BM CD8þCD56þNKT cells of ND-MM (mean ¼ 14.3 � 2.6)and RR-MM (mean¼ 14.1� 2.2) than normal NKT cells (mean¼24.7� 1.1; Fig. 1B, bottom right). Increased expression of PD-1 isdemonstrated by histogram of PD-1 coexpressing effector cells(CD8T cells andNK) in RR-MM(Fig. 1B, right). Similar changes inPD-1 expression were observed in effector cells in the PBMC ofND-MM and RR-MM (data not shown).

We have recently characterized the neutrophil-like CD11bþ

CD14�HLA-DRlow/�CD33þCD15þ MDSC population withtumor-promoting and immune-suppressive activity in the mul-tiple myeloma BM (12). To determine whether MDSC-inducedmultiple myeloma cell growth and immune effector cell sup-pression are mediated by the PD-1/PD-L1 pathway, we nextassessed cell surface expression and frequency of PD-L1 in theBM MDSC of patients with ND-MM and RR-MM. BecauseMDSCs are either absent or present at a very low numbers inhealthy donors, we first evaluated the frequency of PD-L1expressing within myeloid cell subpopulations in ND-MM andRR-MM (Fig. 1C). Myeloid cell subpopulations in each multi-ple myeloma patient BM were phenotypically characterized asCD11bþCD14þHLA-DRþ APCs, CD11bþCD14þHLA-DRlow/�

monocytic myeloid-derived suppressor cells (mMDSC), andCD11bþCD14�HLA-DRlow/�CD33þCD15þ neutrophilic mye-loid-derived suppressor cells (nMDSC). Multiparameter flow-cytometric analysis showed that PD-L1 expression wasincreased in nMDSC (mean ¼ 37.8 � 18) compared with APCs(mean ¼ 21.5 � 7.9) in the BM of patients with ND-MM (Fig.1C, left). Of note, there was a more significant increase in PD-L1 in mMDSC (mean ¼ 33.9 � 10) and nMDSC (mean ¼ 40.1� 11.5) compared with APC (mean ¼ 21.8 � 6) in the BM ofpatients with RR-MM. PD-L1–expressing mMDSC increased inRR-MM (mean ¼ 33.9 � 10) versus ND-MM (mean ¼ 22 �9.4), but there was no significant change in PD-L1–expressingnMDSC with disease progression (Fig. 1C, right). Shown is arepresentative histogram of PD-L1 expression in mMDSC andnMDSC of RR-MM and HD (Fig. 1C, right panel). Thus, PD-L1in multiple myeloma cells and MDSC, along with PD-1 inimmune effector (CD8T cells and NK) cells, were increased inBM of ND-MM and RR-MM.

Checkpoint blockadeovercomesBMstroma-mediatedmultiplemyeloma growth

We next investigated whether PD-1/PD-L1 signaling plays arole in BMSC-mediated multiple myeloma cell growth in cocul-tures ofmultiplemyeloma cells and BMSC frompatients with RR-MM (Fig. 2). BMSCs were generated from multiple myeloma BMand cultured either with multiple myeloma cell lines or autolo-gous CD138þmultiple myeloma cells with or without anti–PD-1and anti–PD-L1, alone or in combination. Multiple myeloma cellviability/growth was measured by CFSE-flow cytometry analysisor 3H-thymidine cell proliferation assays. We first determinedwhether BMSC affects expression of PD-L1 onmultiple myelomacells. BMSC significantly induced PD-L1 expression on H929multiple myeloma cells (>2-fold increase), as shown by multi-parameter flow-cytometric analysis (Fig. 2A). The regulatory roleof PD-1/PD-L1 in BMSC-mediated multiple myeloma growth isfurther evidenced in the cocultures of CFSE-labeled CD138þ

multiplemyeloma cells fromRR-MMwith BMSC,with orwithoutsingle and dual PD-1/PD-L1 signaling blockade. BMSC signifi-cantly increased CD138þCFSElow multiple myeloma cell viabil-ity/growth (CD138þCFSElow cells:80% of CD138þ cells in BM)compared with multiple myeloma cells alone (CD138þCFSElow

cells:2% of CD138þ cells in BM); and blockade of PD-L1(CD138þCFSElow cells:69% of CD138þ cells in BM), PD-1(CD138þCFSElow cells:77% of CD138þ cells in BM), or thecombination (CD138þCFSElow cells:62%of CD138þ cells in BM)overcame BMSC-mediated multiple myeloma cell growth(Fig. 2B). Therefore, the PD-1/PD-L1 pathway may play animportant role in stroma-mediated tumor growth, independentof immune effector cell function.

Checkpoint blockade enhances immune effector cell–mediatedanti–multiple myeloma response in multiple myeloma BM

Wenext investigated the immune-suppressive role of PD-1/PD-L1 signaling in the multiple myeloma microenvironment,and determined whether blockade of PD-1/PD-L1 signalingcan reverse tumor-induced immune suppression in the multi-ple myeloma BM microenvironment (Fig. 3). Immune effectorcell–mediated multiple myeloma cytotoxicity was measured byCFSE/PI apoptotic/dead cell detection assays in coculturesof effector cells (autologous T cells and NK cells) and targetcells (CD138þmultiple myeloma cells) from RR-MM. CD3þTcells, CD56þNK cells, and CD138þ multiple myeloma cellswere isolated by FACS-sorting from BM of patients with RR-MM. CD138þ target multiple myeloma cells were then labeledwith CFSE and cocultured for 4 hours with each autologouseffector cell population in the absence or presence of anti–PD-1and anti–PD-L1, alone or in combination. Apoptotic/deadCD138þ multiple myeloma cells were characterized asCD138þCFSEþPIþ multiple myeloma cells using CFSE/PI-flowcytometry analysis. Blockade of PD-1 and PD-L1, alone andmore significantly in combination, induced effector cell–medi-ated multiple myeloma cytotoxicity. Within the effector cellpopulations, NK cells (4-fold, P < 0.05) demonstrated morepronounced anti–multiple myeloma cytotoxicity than T cells

(Continued.) Gated boxes demonstrate percent intracellular IFNg expression in T cells cultured alone (top), withmMDSC (middle), andwith nMDSC (bottom)with orwithout dual PD-1 and PD-L1 blockade. C, impact of checkpoint blockadewith lenalidomide onMDSC-mediated immune suppression is shown in RR-MMBMby flow-cytometric intracellular cytokine analysis. A representative bar graph shows percent intracellular Gzm-B expression in all effector cells cultured alone or withautologous mMDSC and nMDSC (top). A representative bar graph of intracellular Gzm-B expression is shown in each effector cell population from the coculture ofeffector cells with autologous mMDSC (middle) and with autologous nMDSC (bottom). � , P < 0.05; �� , P < 0.05.







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(2-fold increase, P < 0.05) with blockade of PD-1 (2-fold,P < 0.05) and PD-L1 (3-fold, P < 0.05) alone, or in combination(4-fold, P < 0.05; Fig. 3A).

To define the impact of checkpoint blockade on effector cyto-kine pattern mediating cytotoxicity, we next evaluated intracel-lular production of effector cytokines (IFNg and Gzm-B) in CD4Tcells, CD8T cells, and NK cells cocultured with autologousCD138þ multiple myeloma cells from RR-MM BM (Fig. 3B andC). Intracellular cytokine analysis by multiparameter flow cyto-metry demonstrated that checkpoint blockade induced higherIFNg production in NK cells (anti–PD-L1:1.3-fold, anti–PD-1:1.7-fold, anti–PD-1/PD-L1: 2.2-fold) than T cells during anti–multiple myeloma cytotoxicity (Fig. 3B), along with increasedproduction ofGzm-B (Fig. 3C). Even though checkpoint blockademodulates effector cytokine production, there was no associatedchange in effector cell proliferation (data not shown), indicatingthat checkpoint molecules regulate effector cell function. Of note,checkpoint blockade-induced cell activation varies in each effec-tor cell subpopulation, indicating a differential antitumorresponse in patients.

Checkpoint blockade partially reverses MDSC-mediatedmultiple myeloma growth and immune suppression inmultiple myeloma BM

Because both mMDSC and nMDSC express PD-L1 in RR-MMBM, we next assessed whether blockade of PD-1/PD-L1 signalingcan reverseMDSC-mediated tumor growth and immune suppres-sion in multiple myeloma BM (Fig. 4). The impact of checkpointblockade on MDSC-mediated multiple myeloma growth wasanalyzed in autologous cocultures of CD138þmultiple myelomacells and mMDSC and nMDSC from RR-MM BM. CD138þ

multiplemyeloma cells, CD11bþCD14þHLA-DRlow/negmMDSC,and CD11bþCD14�HLA-DRlow/negCD33þCD15þ nMDSCs wereisolated fromRR-MMBMbyFACS-sorting. CFSE-labeledCD138þ

multiple myeloma cells were cultured for 2 days with autologousmMDSC or nMDSC with or without anti–PD-1 and anti–PD-L1,alone or in combination. CD138þCFSElow multiple myelomacells were assessed by CFSE-flow analysis. As shown in Fig. 4A,mMDSC induced CD138þ multiple myeloma cell growth (21%to 34% CD138þCFSElow cells of CD138þmultiple myelomacells); importantly, dual blockade of PD-1 and PD-L1 abrogatedMDSC-mediated multiple myeloma cell growth in RR-MM BM(34%–22% CD138þCFSElow cells of CD138þ multiple myelomacells). In contrast, there was no significant change in MDSC-mediated multiple myeloma growth by either PD1 or PD-L1single blockade (data not shown).

The impact of checkpoint blockade on MDSC-mediatedimmune suppression was next assessed in RR-MM BM. mMDSC,nMDSC, and autologous effector cells were isolated by FACS-sorting from RR-MM BM. MDSC and autologous effector cells (Tcells, NK cells, and NKT cells) were cultured with or without anti–

PD-1 and anti–PD-L1, alone or in combination, and intracellularproduction of cytokines IFNg and Gzm-B was determined ineffector cells (Fig. 4B and C). As shown by representative multi-parameter dot plots of intracellular IFNg expression in gatedCD3T cells, both mMDSC and nMDSC significantly suppressedIFNg production in T cells, and only combined blockade of PD-1and PD-L1 overcame this suppression (Fig. 4B).

We next tested whether targeting PD-1/PD-L1 inhibitorysignaling using checkpoint blockade antibodies while inducingimmune effector cell activity with lenalidomide can reverseMDSC-mediated immune-suppression in RR-MM BM (Fig. 4C).Intracellular cytokine analysis in effector cells cultured witheither autologous mMDSC or nMDSC demonstrated that bothmMDSC and nMDSC induced suppression of intracellularGzm-B production in all effector cells (Fig. 4C, top). Specifi-cally, checkpoint blockade in cocultures of mMDSC and autol-ogous effector cells induced Gzm-B production in CD8T cells,NK, and NKT cells; and the combination of lenalidomide withcheckpoint blockade further enhanced Gzm-B production, par-ticularly in CD8T cells, NK, and NKT cells (Fig. 4C, middle).Similarly, checkpoint blockade in cocultures of nMDSC withautologous effector cells from RR-MM BM significantlyincreased intracellular Gzm-B production in T cells and NKTcells, but not NK cells (Fig. 4C, bottom). Of note, checkpointblockade, either alone or with lenalidomide, was not able toenhance effector cell proliferation in the presence of MDSC(data not shown).

Lenalidomide reduces expression of PD-1 and PD-L1 in BMcells and enhances checkpoint blockade-induced multiplemyeloma cytotoxicity

The impact of lenalidomide on surface expression of PD-1and PD-L1 in multiple myeloma cells and BM accessory cellswas next defined in RR-MM BM. BMMCs from patients with RR-MM were cultured with lenalidomide (1 mmol/L); and cellsurface expression of PD-1 in effector cells (CD4T cells, CD8Tcells, NK cells, and NKT cells), as well as surface expression ofPD-L1 in CD138þmultiple myeloma cells, MDSC, and CD14þ

monocytes/macrophages, was then determined by multiparam-eter flow cytometry analysis (Fig. 5). Lenalidomide significantlyreduced PD-1 surface expression on CD4T cells, CD8T cells,and NK cells in RR-MM BM (Fig. 5A). Lenalidomide modestlydecreased surface expression of PD-L1 on CD138þ multiplemyeloma cells (Fig. 5B), and more significantly downregulatedPD-L1 expression on monocytes/macrophages and mMDSC inthe BM from RR-MM (Fig. 5C).

Because lenalidomide downregulates surface expression ofcheckpoint molecules in multiple myeloma cells and accessorycells in multiple myeloma BM, we next investigated anti–multiple myeloma cytotoxic activity of lenalidomide in

Figure 5.Lenalidomide reduces expression of PD-1 and PD-L1 and enhances checkpoint blockade-induced multiple myeloma cytotoxicity in multiple myeloma BM. Impact oflenalidomide on cell surface expression of PD-1 on effector cells (A) and PD-L1 on CD138þmultiplemyeloma cells (B), aswell as CD14þmyeloid cells andMDSCs (C) inRR-MM BM is shown by representative histogram plots. The percentage of PD-1 expression on the effector cells and PD-L1 on the CD14þ myeloid cells andMDSCs of untreated BM cells (blue) and lenalidomide-treated BM cells (red) is shown relative to control (gray). D, impact of lenalidomide on checkpoint blockade-inducedmultiplemyeloma cytotoxicity is shown in a representative graph of RR-MMBM.Mononuclear cells frompatientwith RR-MMBMwere labeledwith CFSE andcultured in the absence or presence of anti–PD-1 and anti–PD-L1, alone or in combination, and with or without addition of lenalidomide. Shown is a percentage ofapoptotic/dead (CD138þCFSEþPIþ) multiple myeloma cells in BMMC (top). Impact of lenalidomide on checkpoint blockade-induced effector cell activity isshown in RR-MM BM by intracellular effector cytokine analysis. RR-MM BM cells were cultured with anti–PD-1 and anti–PD-L1 alone, or in combination, or with theaddition of lenalidomide. Representative bar graph shows intracellular expression of IFNg in each effector cell population in RR-MM BM (bottom). � , P < 0.05.






combination with checkpoint blockade in RR-MM (Fig. 5D). Tomimic the BM microenvironment, all BM cells were labeledwith CFSE and cultured with anti–PD-1 anti–PD-L1, alone ortogether, and with lenalidomide. Apoptotic/dead CD138þ

CFSEþPIþ multiple myeloma cells were identified by multipa-rameter CFSE/PI-flow cytometry analysis. There was a signifi-cant increase in CFSEþPIþ apoptotic/dead CD138þ multiplemyeloma cells in BM cultured with anti–PD-1 and anti–PD-L1;and lenalidomide further enhanced checkpoint blockade-medi-ated multiple myeloma cytotoxicity in RR-MM BM (Fig. 5D,top). We further analyzed the effect of checkpoint blockade,alone or with lenalidomide, on cytokine production in BMeffector cells. BM cells were cultured with anti–PD-1, anti–PD-L1, or anti–PD-1/PD-L1, alone or with lenalidomide. Intracel-lular expression of effector cytokines (IFNg and Gzm-B) wasthen measured in CD4T cells, CD8T cells, NK cells, NKT cells,and monocytes/macrophages by multiparameter flow-cyto-metric analysis (Fig. 5D, bottom). Intracellular cytokine anal-ysis of effector cells in RR-MM BM demonstrated that dualblockade of PD-1 and PD-L1 significantly induced IFNg pro-duction in all effector cells (Fig. 5D, bottom); as well as Gzm-Bproduction in NK cells and NKT cells (data not shown).Importantly, lenalidomide further enhanced dual checkpointblockade-induced IFNg production in all effector cells (Fig. 5D,bottom).

DiscussionThe multiple myeloma microenvironment is transformed in

the presence of tumor cells to promote multiple myeloma devel-opment/growth while allowing tumor escape from immunesurveillance due to suppression of anti–multiple myelomaimmune effector responses. As a result, infections remain a majorcause of death (28). Recent studies have defined immune check-point receptor PD-1/PD-L1 signaling as a key pathway regulatingthe critical balance between immune activation and tolerance(23, 28–31). Binding of PD-1 on effector cells to PD-L1 or PD-L2on non-hematopoietic cells triggers inhibitory signaling in effec-tor cells, leading to induction andmaintenance of tolerance (32).Recent studies in solid tumors have demonstrated that PD-1/PD-L1 signaling allows for escape from immune surveillance, trans-forming the tumor microenvironment into a tumor-protective,immune-suppressive milieu (7, 8, 16, 18, 33–36). Specifically,PD-L1 expression on tumor cells inhibits T-cell activationand CTL-mediated tumor lysis. Importantly, recent studieshave shown increased expression of PD-L1 on lung, skin, renal,gastric, pancreatic, colorectal, breast, and ovarian cancers (8, 15,16, 20, 35, 37–40). Moreover, blockade of PD-1/PD-L1 signalingusing clinically relevant anti–PD-1 monoclonal antibodiesrestored immune responses and achieved remarkable clinicalresponses in solid tumors, including melanoma and lung cancer,providing a very promising novel immunotherapeutic strategy.

Studies in hematologic malignancies have shown increasedexpression of PD-L1 in B-cell lymphomas, chronic lymphocyticleukemia, acute myeloid leukemia, and multiple myeloma(24, 25, 33, 41–45). In the present study, we investigated therole of PD-1/PD-L1 inhibitory signaling in the bidirectionalinteraction between tumor, stroma, and immune accessory cellsin themultiplemyeloma BMmicroenvironment. Importantly, weassessed the impact of single and dual blockade of PD-1/PD-L1signaling, alone or in combination with lenalidomide, on

the tumor-promoting, immune-suppressive multiple myelomamicroenvironment. Previous studies have demonstrated that PD-L1 is not expressed on normal plasma cells, but is expressed onmultiple myeloma cell lines and primary multiple myeloma cells(25–27). Here, we showed that both mRNA and cell surfaceexpression of PD-L1 is increased on CD138þ multiple myelomacells from ND-MM and further elevated in RR-MM, comparedwith normal BM plasma cells. However, within a broad panel ofmultiple myeloma cell lines, constitutive PD-L1 expression islimited to MM1.S, OPM2, and H929 cells, suggesting that PD-L1 expression is induced on multiple myeloma cells by thebidirectional interaction between tumor and accessory cells.PD-L1 gene is encoded on chromosome 9, which is increased incopy number in HMM. Importantly, patients with HMM have abetter prognosis and outcome than patients with NHMM. Anal-ysis of PD-L1 gene expression in tumor cells from patients withND-MM demonstrated that the expression of PD-L1 gene wassignificantly correlated with the copy number in most patients'tumor clones. Although expression of PD-L1 gene was signifi-cantly increased in NHMM relative to normal donor plasmacells, it was even higher in HMM. Enhancing anti–multiplemyeloma immune response by targeting checkpoint moleculesmay therefore improve outcome even in HMM.

PD-1 expression is increased on CD4T cells from patients,and returns to levels in normal CD4T cells following autolo-gous transplant (25). Benson and colleagues (22). have shownthat PD-1 is expressed on NK cells from multiple myelomapatients, but not normal NK cells; that blockade of PD-1signaling by anti–PD-1 antibody induces cytolytic activity ofNK cells; and that lenalidomide further induces NK-mediatedantitumor responses. Here, we extended these studies to deter-mine the impact not only of PD-1 blockade but also of dualblockade of PD-1 and PD-L1, alone or with lenalidomide, onthe functional sequelae in multiple myeloma cells, stroma,immune effector cells (CD4T cells, CD8T cells, NK cells, NKTcells, and monocytes/macrophages) and immune suppressorMDSCs in the multiple myeloma BM.

We first determined that PD-1 expression is significantlyincreased on effector immune cells, particularly on CD8T cellsandNK cells, whereas PD-L1 is expressed bymyeloid effector cellsmonocytes/macrophages. Previous studies have shown thatMDSCs express high levels of B7 inmurinemodels of solid tumor(46–48). Moreover, It has been recently demonstrated that PD-1and PD-L1 are expressed at low levels in MDSC of patients withmultiplemyeloma (49). Here, we compared PD-L1 expression onmyeloid cell subpopulations, including APCs, mMDSC, andnMDSC in the BM of patients with multiple myeloma. PD-L1expression is significantly higher in MDSC in RR-MM than ND-MM. Increased expression of PD-1 on immune effector cells, andincreased PD-L1 on both multiple myeloma cells and immunesuppressor MDSC, indicate that PD-1/PD-L1 inhibitory signalingplays an important role in providing a tumor-promoting,immune-suppressive microenvironment in multiple myelomaBM.

Extensive studies focusing on the interaction of BMSC withmultiple myeloma cells have demonstrated that BMSCs pro-mote multiple myeloma cell growth and drug resistance.Tamura and colleagues (26) have demonstrated that BMSCalso upregulates PD-L1 expression on multiple myeloma cells.To delineate whether PD-1/PD-L1 plays a role in BMSC-medi-ated multiple myeloma growth, we assessed the impact of


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single and dual blockade of PD-1/PD-L1 signaling in coculturesof tumor cells from patients with RR-MM and BMSC. Impor-tantly, BMSC markedly induced PD-L1 expression in multiplemyeloma cells, and BMSC-mediated multiple myeloma cellgrowth was abrogated by blockade of PD-1 and PD-L1, suggest-ing that checkpoint blockade may have a direct effect on BMSC-induced multiple myeloma growth, independent of its immuneaccessory cell activity.

Immunomodulatory drug lenalidomide not only targets themultiple myeloma cell directly, but also induces anti–multiplemyeloma activity of immune effector cells. We have recentlyshown that lenalidomide does not alter MDSC-mediated tumorgrowth and immune suppression in multiple myeloma (14).However, lenalidomide reduces PD-1 expression on NK cells andPD-L1 expression on tumor cells from patients with multiplemyeloma (22). Here, we found that lenalidomide decreased PD-1expression in all effector cells (CD4T cells, CD8T cells, NK cells,and NKT cells), as well as PD-L1 expression in multiple myelomacells, MDSC, and monocytes/macrophages. We characterized theimmunomodulatory effects of PD-1/PD-L1 blockade with lena-lidomide in autologous cocultures of immune effector cells withmultiple myeloma cells from patients with RR-MM. Even thoughthere was no change in effector cell proliferation, PD-1/PD-L1blockade significantly induced cytotoxic activity of autologous Tcells, NK cells, and monocytes/macrophages against multiplemyeloma cells; and lenalidomide further enhanced effectorcell–mediated cytotoxicity. PD-1/PD-L1 blockade also inducedintracellular expression of cytotoxic cytokines IFNg and Gzm-B inCD4T cells, CD8T cells, NK cells, andmonocytes/macrophages inRR-MM. Furthermore, MDSC-mediated multiple myeloma cellgrowth was significantly decreased by PD-1/PD-L1 blockade.Finally, PD-1/PD-L1 blockade induced intracellular expressionof IFNg and Gzm-B in T cells, NK cells, and NKT cells culturedwith autologous MDSC; and lenalidomide further enhanced thiseffector cell activation. Of note, checkpoint blockade inducedresponse in each effector cell population regardless of PD-1expression level.

Our data therefore demonstrate that immune checkpointsignaling plays an important role conferring the tumor-pro-moting, immune-suppressive microenvironment in multiplemyeloma BM. Importantly, blockade of PD-1 or PD-L1, aloneand in combination, induces anti–multiple myeloma immuneresponses, which can be further enhanced by lenalidomide.

Targeting checkpoint signaling using PD-1 and PD-L1–blockingantibodies, particularly in combination with lenalidomide,therefore represents a promising novel immune-based thera-peutic strategy to inhibit tumor cell growth, restore hostimmune function in multiple myeloma, and improve patientoutcome in multiple myeloma.

Disclosure of Potential Conflicts of InterestJ.P. Laubach reports receiving commercial research grants from Celgene,

Millennium, Novartis, and Onyx. N.C. Munshi, P.G. Richardson, and K.C.Anderson are consultants/advisory board members for Celgene. No potentialconflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: G. G€org€un, T. Hideshima, K.C. AndersonDevelopment of methodology: G. G€org€unAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): G. G€org€un, K.B. Cowens, S. Paula, G. Bianchi,R. Suzuki, Y.-T. Tai, F. Magrangeas, S. Minvielle, H. Avet-Loiseau, N.C. Munshi,D.M. Dorfman, P.G. Richardson, K.C. AndersonAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): G. G€org€un, M.K. Samur, H. Ohguchi, S. Kikuchi,T. Harada, P.G. Richardson, K.C. AndersonWriting, review, and/or revision of the manuscript:G. G€org€un, J.E. Anderson,J.P. Laubach, N. Raje, N.C. Munshi, D.M. Dorfman, P.G. Richardson,K.C. AndersonAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): G. G€org€un, S. Paula, H. Avet-LoiseauStudy supervision: K.C. AndersonOther (sample collection): R.E. WhiteOther (research assistance/observation): A. Singh

Grant SupportThis work was supported by an NIH/NCI Specialized Program of Research

Excellence in Myeloma P50 CA100707 (to K.C. Anderson); NIH/NCI Host–Tumor Cell Interactions in Myeloma: Therapeutic Applications P01 CA78378(to K.C. Anderson), and NIH/NCI Molecular Sequelae of Myeloma–BoneMarrow Interactions: Therapeutic Applications R01 CA50947 (to K.C. Ander-son) grants.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received January 26, 2015; revised April 10, 2015; accepted May 1, 2015;published OnlineFirst May 15, 2015.

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2015;21:4607-4618. Published OnlineFirst May 15, 2015.Clin Cancer Res Güllü Görgün, Mehmet K. Samur, Kristen B. Cowens, et al. Immune Response in Multiple MyelomaLenalidomide Enhances Immune Checkpoint Blockade-Induced

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